Peptide substrates, method of preparation and use in the determination of protein C

ABSTRACT

The present invention relates, by way of novel industrial products, to peptide compounds selected from the group consisting of 
     (i) the tripeptides of the formula 
     
         Y--A.sub.1 --A.sub.2 --A.sub.3 --R                         (I) 
    
      in which 
     Y is H or an appropriate group blocking the N-terminal end, 
     A 1  is an amino acid residue selected from the group consisting of (2-oxothiazolidin-4-yl)carbonyl [abbreviated to THC], (2-oxotetrahydro-1,3-thiazin-5yl)carbonyl [abbreviated to TZC], thiazolidine-4-carbonyl [abbreviated to ATC] and (tetrahydro-1,3-thiazin-5-yl)carbonyl [abbreviated to AZC] residues, 
     A 2  is an amino acid residue selected from the group consisting of Pro, 3Hyp and 4Hyp residues, where the OH side-group of 3Hyp and 4Hyp is capable of being protected by an ether or ester protecting group, 
     A 3  is an amino acid residue selected from the group consisting of Arg and Lys residues, and 
     R is a labeling means; and 
     (ii) their addition salts. 
     These novel compounds are useful as substrates in the determination of Protein C.

FIELD OF THE INVENTION

The present invention relates, by way of novel industrial products, tothe peptide substrates of formula I below and their addition salts.

It further relates to the method of preparing these novel products andto their use in the field of the determination of Protein C.

PRIOR ART

It is known that a number of peptide substrates have already beenproposed in the past for the identification and assay of numeroussubstances involved in the cascade mechanism of hemostasis.

It is known in particular that the substrate

    H--L--Pyr--L--Pro--L--Arg--pNA                             (IIa)

[in which Pyr is the pyroglutaminyl residue (i.e. 5-oxoprolyl orpyrrolidin-2-one-5-carbonyl) and pNA is the p-nitroanilino radical],described in EP-A-0 004 256 as a chromogenic substrate for thedetermination of serine proteases and SH-proteases, has recently provedeffective in the determination of Protein C.

It is also known that the substrate

    H--D--Lys(Cbo)--L--Pro--L--Arg--pNA                        (IIb)

has already been used in the field of the determination of Protein C,but that its efficacy is inferior to that of the aforementioned compoundIIa.

There is therefore a need for specific peptide substrates for thedetermination of Protein C. In fact, with the exception of theafore-mentioned substrates IIa and IIb, there are no other specificsubstrates for Protein C which are commercially available.

To meet this need, a novel technical solution is proposed, according tothe invention, which uses peptide substrates having a differentstructure from that of the afore-mentioned products of formulae IIa andIIb known in the prior art.

SUMMARY OF THE INVENTION

According to a first feature of the invention, peptide substrates areprovided, as novel industrial products, which are selected from thegroup consisting of

(i) the tripeptides of the formula

    Y--A.sub.1 --A.sub.2 --A.sub.3 --R                         (I)

in which

Y is H or an appropriate group blocking the N-terminal end,

A₁ is an amino acid residue selected from the group consisting of(2-oxothiazolidin-4-yl)carbonyl [abbreviated to THC],(2-oxotetrahydro-l,3-thiazin-5-yl)carbonyl [abbreviated to TZC],thiazolidine-4-carbonyl [abbreviated to ATC] and(tetrahydro-1,3-thiazin-5-yl)carbonyl [abbreviated to AZC] residues,

A₂ is an amino acid residue selected from the group consisting of Pro,3Hyp and 4Hyp residues, where the OH side-group of 3Hyp and 4Hyp iscapable of being protected by an ether or ester protecting group,

A₃ is an amino acid residue selected from the group consisting of Argand Lys residues, and

R is a labeling means; and

(ii) their addition salts.

According to a second feature of the invention, the tripeptide compoundsof formula I and their addition salts are used for the determination ofProtein C. To this end, a method of determination for the assay oridentification of Protein C is provided which comprises bringing atripeptide of formula I or one of its addition salts into contact with abody fluid (especially plasma, blood serum, or urine) or any othersample of natural or synthetic origin which may contain Protein C.

Finally, according to a third feature of the invention, a method ofpreparing the tripeptide compounds of formula I and their addition saltsis provided.

ABBREVIATIONS

For convenience, the following abbreviations have been used in thepresent invention:

    ______________________________________                                        the amino acid residues:                                                      Arg =     arginyl                                                             ATC =     thiazolidine-4-carbonyl (or thioprolyl)                             AZC =     (tetrahydro-1,3-thiazin-5-yl)carbonyl                               3Hyp =    3-hydroxypropyl (or 3-hydroxypyrrolidine-2-                                   carbonyl)                                                           4Hyp =    4-hydroxypropyl (or 4-hydroxypyrrolidine-2-                                   carbonyl)                                                           Lys =     lysyl                                                               Pro =     prolyl                                                              Pyr =     pyroglutaminyl (or pyrrolidin-2-one-5-                                        carbonyl)                                                           THC =     2-oxothiazolidin-4-yl)carbonyl                                      TZC =     (2-oxotetrahydro-1,3-thiazin-5-yl)carbonyl                          the other abbreviations:                                                      Ac =      acetyl                                                              AcOH =    acetic acid                                                         Adoc =    adamantyloxycarbonyl                                                Aoc =     t-amyloxycarbonyl                                                   Boc =     t-butoxycarbonyl                                                    Bop =     (benzotriazol-1-yl)oxytris(dimethylamino)-                                    phosphonium hexafluorophosphate (alternative                                  nomenclature: CASTRO's reagent) of the for-                                   mula                                                                           ##STR1##                                                           Bu =      n-butyl                                                             Bz =      benzoyl                                                             Bzl =     benzyl                                                              Cbo =     carbobenzoxy                                                        o-Cl-pNA =                                                                              o-chloro-p-nitroanilino [or (2-Cl)pNA]                              DCCI =    dicyclohexylcarbodiimide                                            DIEA =    diisopropylethylamine                                               DMF =     dimethylformamide                                                   Et =      ethyl                                                               Et.sub.3 N =                                                                            triethylamine                                                       EtO =     ethoxy                                                              Fmoc =    fluoren-9-ylmethoxycarbonyl                                         Foc =     furfuryloxycarbonyl                                                 HMPT =    N,N,N',N',N",N"-hexamethylphosphorotri-                                       amide                                                               HOBT =    1-hydroxybenzotriazole                                              HPLC =    high performance liquid chromatography                              H-TFA =   trifluoroacetic acid (or HTFA)                                      Iboc =    isobornyloxycarbonyl                                                iPr =     isopropyl                                                           Me =      methyl                                                              MW =      molecular weight                                                    OD =      optical density                                                     Ph =      phenyl                                                              pH =      cologarithm of the concentration of H.sup.+  ions                   pNA =     p-nitroanilino [or (4-NO.sub.2)C.sub.6 H.sub.4 NH]                  Pr =      n-propyl                                                            RT =      room temperature (15-20° C.)                                 tBu =     t-butyl                                                             THF =     tetrahydrofuran                                                     TLC =     thin layer chromatography                                           Tos =     p-toluenesulfonyl (or tosyl)                                        Z =       benzyloxycarbonyl                                                   Z(p-Cl) = p-chlorobenzyloxycarbonyl                                           Z(p-OMe) =                                                                              p-methoxybenzyloxycarbonyl                                          ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION

In formula I above, the amino acid residue A₁ can be represented by thefollowing structural formula: ##STR2## in which

B is a single bond or the group CH₂, and

X is CH₂ or CO.

When the pair (B;X) in formula III is (-;CO), (CH₂ ;CO), (-;CH₂) or (CH₂;CH₂), A₁ is respectively THC, TZC, ATC or AZC.

According to the invention, the amino acid residue A₁ will have the D orL configuration or else be a racemic mixture (DL) of these twoconfigurations. A₁ will preferably be D-THC, D-TZC, D-ATC and D-AZC andmore preferably L-THC, L-TZC, L-ATC and L-AZC. The amino acid residue A₁which is considered to be the most valuable according to the inventionis L-THC.

The hydrogen atom and radicals blocking the N-terminal end of peptides,such as those described in EP-A-0 110 306, U.S. application No. 4 480030, FR-A-2 293 439 and U.S. application No. 4 440 678, may be mentionedamong the groups Y which are suitable. The following may be indicated inparticular among the groups Y, other than H, blocking said N-terminalend: C₁ -C₄ alkyl groups (especially Me, Et, Pr, iPr, Bu, tBu),substituted or unsubstituted aryl groups (especially Ph, tolyl, xylyl,chlorophenyl, trifluoromethylphenyl, methoxyphenyl), substituted orunsubstituted aralkyl groups (especially Bzl, chlorobenzyl,dichlorobenzyl, trifluoromethylbenzyl, difluorobenzyl, methoxybenzyl,ethoxybenzyl, 3,4-methylenedioxybenzyl) and conventional protectinggroups for the N-terminal end of peptides [especially Ac, Tos, Adoc,Aoc, Bz, Cbo, Fmoc, Foc, Iboc, Z, Z(p-Cl) and Z(p-OMe)].

The group Y other than H is introduced into the tripeptide of formula Iin accordance with one of the following two reaction schemes: ##STR3##in which the OH group (of the carboxylic acid group) bonded to theCO-terminal end of A₁ is protected if appropriate; and ##STR4##

The choice of scheme A or B will depend on (i) the nature of theblocking group, and (ii) the labile character of the hydrogen atom ofthe N-terminal end which is located in the 3 position of the ring of A₁.With this in mind, scheme A will preferably be used when A₁ is THC orTZC.

Furthermore, as the rings of THC and AZC are generally obtained bycyclization at elevated temperature, the cyclization can be perturbedand/or the yields reduced by the presence of a group Y other than H,especially Y=alkyl, aryl or aralkyl, in the pyrolysis reactionmechanisms: ##STR5## It is therefore advantageous to prepare the acidsH-THC-OH and H-AZC-OH by pyrolysis and then to replace the H atom of theN-terminal end in accordance with the afore-mentioned scheme A to givethe compounds of formula I in which Y is other than H.

The preferred group Y according to the invention is H. Briefly, thegroup Y=H will be replaced with a group Y other than H if and only ifsuch a replacement substantially enhances the specificity of thesubstrate Y--A₁ --A₂ --A₃ --R towards Protein C.

As indicated above, the OH side-group of 3Hyp and 4Hyp can be protectedin the form of an ether or ester. The protecting group replacing the Hatom of said OH group may be in particular a C₁ -C₄ alkyl group(especially Me, Et, Pr, iPr, Bu, tBu), a substituted or unsubstitutedaryl group (especially Ph, tolyl, xylyl, chlorophenyl,trifluoromethylphenyl, methoxyphenyl) or a substituted or unsubstitutedaralkyl group (especially Bzl, chlorobenzyl, difluorobenzyl,trifluoromethylbenzyl, dichlorobenzyl, methoxybenzyl, ethoxybenzyl,3,4-methylenedioxybenzyl), as envisaged in the definition of Y, in thecontext of protection in the form of an ether, or a C₂ -C₅ aliphaticacyl group (especially Ac, propionyl, butanoyl, pentanoyl) or anaromatic group (especially Bz, toluoyl, chlorobenzoyl, methoxybenzoyl,3,4-methylenedioxybenzoyl) in the context of protection in the form ofan ester. If appropriate, said H atom may also be replaced with Tos or atrialkylsilyl group (especially trimethylsilyl or triethylsilyl) or thelike.

Advantageously, the amino acid residues A₂ and A₃ will have the Lconfiguration and the preferred groups for A₂ and A₃ according to theinvention will be L-Pro, L-3Hyp and L-4Hyp and, respectively, L-Arg andL-Lys.

The labeling means R is well known in the art of biological andmicrobiological assays; reference is made in this connection to theprior art cited above and especially document U.S. Pat. No. 4,448,715.Said labeling means will preferably be selected from the groupconsisting of aminated groups NH-R' which (i) induce a color change,(ii) induce a change in fluorescence, or (iii) contain at least oneradioactive element (for example an anilino or benzylamino group labeledwith a ¹⁴ C or ³ H radioisotope). Any amino group NH-R' which gives,during or after the enzymic reaction, a signal capable of beingamplified for detection (for example by measurement of the opticaldensity at a given wavelength, or by measurement of the radioactivity)is suitable for the purposes of the invention. The amount of product H-Robtained by cleavage in the enzymic hydrolysis is proportional to theamount of enzyme used. Said amount of H-R can be determined byphotometry, spectrophotometry, fluorospectrophotometry orelectrochemistry.

The group R which is preferred according to the invention is achromogenic group, typically a nitrophenylamino group (in which thephenyl radical is capable of being substituted by a group COOH, F, Cl,Br, CH₃, OCH₃, CN, CF₃ and/or SO₃ H), or a fluorogenic group, typicallya naphthylamino group (in which the naphthyl radical is capable of beingsubstituted by a group OCH₃, COOH, SO₃ H or CH₃), and4-methylcoumaryl-7-amino, 4-trifluoromethylcoumaryl-7-amino andanalogous groups.

The following may be mentioned in particular among the chromogenic andfluorogenic aminated groups which are suitable according to theinvention: p-nitro-anilino (abbreviated to pNA),2-carboxy-4-nitroanilino and 3-carboxy-4-nitroanilino,2-halogeno-4-nitroanilino and 3-halogeno-4-nitroanilino (in which thehalogen is F, C1 or Br), 2-methoxy-5-methyl-4-nitroanilino,2-hydroxysulfonyl-4-nitroanilino, 4-trifluoromethyl-2-nitroanilino,4-trifluoromethyl-3-nitroanilino, 4-cyano-2-nitroanilino,naphthyl-2-amino, 4-hydroxysulfonylnaphthyl-1-amino, quinolylamino,nitroquinolylamino and the like.

The preferred group R according to the invention is a chromogenic group,namely on the one hand pNA and on the other hand analogous groups inwhich the phenyl ring of pNA is substituted in the 2 or 3 position, saidgroups having the formula ##STR6## in which Y' is Br, Cl, F, CF₃, COOH,COOW, CONH₂, CONHW, CONW₂, CONH(CH₂)mNMe₂, OH or OW, in which W is a C₃-C₆ alkyl, C₆ -C₁₀ aryl, C₇ -C₁₁ aralkyl or C₃ -C₈ alicyclic group and mis an integer having a value of 1 to 10.

Such groups of formula VI in which the phenyl ring of the pNA group issubstituted are described especially in document EP-A-0 110 306.

The addition salts according to the invention are essentially acidaddition salts obtained by reacting a compound of formula I with amineral or organic acid.

The best mode of carrying out the invention, which is recommended here,consists in using a substrate selected from the group consisting of

(a) the tripeptide compounds of the formula

    H--A.sub.1 --A.sub.2 --A.sub.3 --pNA                       (I')

in which

A₁ is D-THC, D-TZC, D-ATC, D-AZC, L-THC, L-TZC, L-ATC or L-AZC,

A₂ is L-Pro, L-3Hyp or L-4Hyp and

A₃ is L-Arg or L-Lys; and

(b) their addition salts.

In this best mode of carrying out the invention, it is recommended moreparticularly to use L-TZC, L-ATC or, preferably, L-THC as the amino acidresidue A₁.

For convenience, in the following description, an amino acid residuementioned without the configuration (for example Pro) denotes, unlessindicated otherwise, that said amino acid residue has the Lconfiguration (i.e. L-Pro in the example in question).

Without in any way implying a limitation, a number of peptide compoundsaccording to the invention have been collated in Table I below.

                  TABLE I                                                         ______________________________________                                        H--A.sub.1 --A.sub.2 --A.sub.3 --pNA.HX                                       Compound  A.sub.1    A.sub.2   A.sub.3                                                                            HX                                        ______________________________________                                        Ex. 1     THC        Pro       Arg  H--TFA                                    Ex. 2     THC        Pro       Arg  AcOH                                      Ex. 3     THC        3Hyp      Arg  AcOH                                      Ex. 4     THC        4Hyp      Arg  AcOH                                      Ex. 5     THC        Pro       Lys  AcOH                                      Ex. 6     TZC        Pro       Arg  AcOH                                      Ex. 7     ATC        Pro       Arg  2AcOH                                     Ex. 8     AZC        Pro       Arg  2AcOH                                     CP 1 (a)  Pyr        Pro       Arg  AcOH                                      CP 2 (b)  D-Lys(Cbo) Pro       Arg  2AcOH                                     ______________________________________                                         Notes                                                                         (a) reference product of formula IIa described in EPA-0 004 256               (b) reference product of formula IIb                                     

The tripeptide compounds of formula I and their addition salts accordingto the invention can be prepared in accordance with a method known perse by the application of conventional reaction mechanisms of peptidesynthesis. The method of preparation which is recommended here comprisesreacting 1 mol of a dipeptide of the formula

    H--A.sub.2 --A.sub.3 --R                                   (IV)

in which A₂, A₃ and R are defined as indicated above, with 1.0 to 1.3mol of an amino acid of the formula

    Y--A.sub.3 --T                                             (V)

in which Y and A₃ are defined as indicated above and T is OH, F, Cl orBr.

Advantageously, the reaction IV+V=I is carried out in an inert solvent,especially DMF or THF, at a temperature of between 0° and 50° C.,especially at RT. Again advantageously, the reaction medium willcomprise an excess of a base acting as a cosolvent and as a protonacceptor, such as Et₃ n or DIEA. When said reaction is carried out, acoupling agent, especially Bop or HOBT, will also be incorporated intosaid reaction medium, in particular when T is OH. When HOBT is used asthe coupling agent, an appropriate amount of DCCI will advantageously beassociated with said agent.

In practice, molar ratios IV/Et₃ N or IV/DIEA of 1/2 to 1/4 will be usedand, when T=OH, molar ratios IV/HOBT or IV/Bop of 1/1 to 1/2 will beused; when DCCI is used, the molar ratio IV/DCCI will be of the order of1/1.5-1/2. The purpose of the coupling agents HOBT and Bop is toactivate the carboxylic acid group, COOH, of the CO-terminal end (whenT=OH ) and they have the advantage of not causing racemization. Theagent Bop will be used at a pH of 7-8 and the agent HOBT at a pH of 5 to8. With regard to its coupling properties, Bop will be preferred toHOBT. The tripeptide of formula I is isolated from the reaction mediumof the reaction IV+V=I in accordance with the following steps:

evaporation of the inert solvent to dryness under vacuum,

precipitation of the resulting evaporation residue with ether (MeOMe orEtOEt), followed by recovery of the precipitate by filtration,

taking-up of the filtered precipitate in the minimum amount of a CHCl₃/MeOH/AcOH mixture,

chromatography of the resulting mixture using said solvent CHCl₃/MeOH/AcOH, and

pooling of the homogeneous fractions resulting from said chromatography,and evaporation of said solvent.

The tripeptide compounds of formula I and their addition saltsconstitute specific substrates for Protein C. In the recommended methodof determining Protein C, a given amount of a tripeptide of formula I orof one of its addition salts (at a concentration of the order of 10mg/ml) is brought into contact, in an appropriate aqueous liquid mediumsuch as a buffered isotonic solution, with a test sample (diluted ifappropriate) which may contain Protein C, for at least 0.5 h at atemperature of between RT and 40° C., especially 37° C.

The activity of the tripeptides according to the invention towardsProtein C was determined by one of the conventional methods, such as theone described in publication EP-A-0 280 160 (see page 14), thehydrolysis rate being assessed by the variation in optical density withtime (.increment.OD/min). The results obtained with equimolar doses havebeen collated in Table II below, where the activity of the referenceproduct of formula IIa is specified as being equal to 100% for the sakeof convenience.

                  TABLE II                                                        ______________________________________                                        ACTIVITY TOWARDS PROTEIN C                                                           Product                                                                              Activity                                                        ______________________________________                                               Ex. 1  148%                                                                   Ex. 2  153%                                                                   Ex. 3  118%                                                                   Ex. 4  111%                                                                   Ex. 5  122%                                                                   Ex. 6  105%                                                                   Ex. 7  110%                                                                   Ex. 8  103%                                                                   CP 1   100%                                                                   CP 2    34%                                                            ______________________________________                                    

The comparative results in Table II show that the tripeptides accordingto the invention are at least as active as the reference product CP 1.

An assay kit for the determination of Protein C, which contains atripeptide of formula I or one of its addition salts and, ifappropriate, a standard sample of Protein C (of human or bovine origin)and of buffered dilution media, is also provided according to theinvention.

Further advantages and characteristics of the invention will beunderstood more clearly from the following description of PreparatoryExamples. These data do not in any way imply a limitation and are givenby way of illustration.

PREPARATION I

Preparation of L-THC-L-Pro-L-Arg-pNA.H-TFA (Example 1)

a) Z-L-Arg-pNA.HCl

3.44 g (0.01 mol) of Z-L-Arg-OH.HCl are dissolved in 20 ml of anhydrousHMPT at RT and 1.39 ml (0.01 mol) of Et₃ n are then added at RT, withstirring. 2.46 g (0.015 mol) of p-nitrophenyl isocyanate are added tothe resulting solution. The resulting reaction medium is stirred for 24h at RT and then evaporated under vacuum and the residue is taken upwith the minimum amount of AcOH and then diluted with AcOEt. Theresulting solution is subsequently extracted successively three timeswith small amounts of 0.5 M NaHCO₃, three times with a solution of KHSO₄at 50 g/l and then several times with H₂ O semisaturated with NaCl. Theorganic phase is then dried over anhydrous sodium sulfate. Afterfiltration (removal of Na₂ SO₄), the solvent is evaporated off and theevaporation residue is recrystallized from an AcOEt/MeOMe mixture (3/7v/v) to give 3.5 g of the expected product in the form of a whitepowder. M.p.=128°-130° C.

Analysis (TLC on silica gel):

Rf=0.5 in AcOEt/pyridine/AcOH/H₂ O (20/4.5/3/1 v/v);

Rf=0.69 in CHCl₃ /MeOH/AcOH (5/3/1 v/v).

b) H-L-Arg-pNA,2HBr

1 g (2.15 mmol) of Z-L-Arg-pNA.HC1 is charged into a glass/Teflonapparatus. 8 ml of glacial AcOH, 2 ml of anisole and 10 ml of a solutionof HBr (at 33% w/v) in AcOH are added successively under an inertatmosphere (stream of nitrogen). The reaction is left to proceed for 1 hat RT under a nitrogen atmosphere. After this time has elapsed, thereaction mixture, which has become homogeneous during the deprotection,is precipitated in ether (MeOMe or EtOEt). After decantation, thesupernatant is discarded and the precipitate is washed several timeswith ether. The precipitate is collected by filtration and dried undervacuum over KOH for 24 h to give 0.95 g of the expected product.

Analysis (TLC on silica gel):

Rf=0.04 in AcOEt/pyridine/AcOH/H₂ O (20/4.5/3/1.5 v/v);

Rf=0.38 in BuOH/AcOH/H₂ O (3/1/1 v/v).

c) BOC-L-Pro-L-Arg-pNA.HBr

1 g (2.19 mmol) of H-L-Arg-pNA.2HBr is dissolved in 10 ml of DMF, and0.854 ml (6.57 mmol) of DIEA is then added. In another vessel, asolution of 4.71 mg (2.19 mmol) of Boc-L-Pro-OH in 50 ml of DMF isneutralized with 0.285 ml of DIEA. The two solutions are mixed and 970mg of Bop are then added while the resulting mixture is kept at RT, withstirring, the pH being kept at between 7.0 and 8.0 throughout thereaction by the addition of small portions of DIEA. After one hour, thereaction is complete and the reaction mixture is evaporated to drynessunder vacuum; the evaporation residue is taken up with an AcOEt/MeOHmixture and extracted with a 0.5 M aqueous solution of NaHCO₃. Theorganic phase is dried over sodium sulfate, concentrated andprecipitated in ether (MeOMe or EtOEt) to give 877 mg (yield: 70%) ofthe expected product.

Analysis (TLC on silica gel):

Rf=0.27 in CHCl₃ /MeOH/AcOH (20/3/1 v/v);

Rf=0.71 in CHCl₃ /MeOH/AcOH (10/3/1 v/v).

d) H-L-Pro-L-Arg-pNA.2H-TFA

850 mg (1,485 mmol) of Boc-L-Pro-L-Arg-pNA.HBr and then 6 ml of CH₂ Cl₂and 6 ml of H-TFA are introduced successively into a reactor. After areaction time of 0.25 h, the reaction mixture is precipitated directlyin ether. After drying of the resulting precipitate, 837 mg of theexpected product are obtained.

Analysis (TLC on silica gel):

Rf=0.56 in BuOH/AcOH/H₂ O (3/1/1 v/v);

Rf=0.13 in CHCl₃ /MeOH/AcOH (5/3/1 v/v).

e) L-THC-L-Pro-L-Arg-pNA.H-TFA

Following the operating protocol described in section c) above, areaction mixture comprising (i) 800 mg (1.3 mmol) ofH-L-Pro-L-Arg-pNA.2H-TFA, (ii) 190 mg (1.3 mmol) of H-THC-OH, (iii) 1 mlof DIEA and (iv) 5.75 mg (1.3 mmol) of Bop is reacted in DMF at RT for1.5 h, with stirring. The reaction mixture is subsequently evaporatedunder vacuum and the residue is then precipitated in EtOEt. The whiteprecipitate obtained is taken up in the minimum amount of the solventsystem CHCl₃ /MeOH/AcOH (5/3/1 v/v) and chromatographed on silica gelusing the same solvent system. The homogeneous fractions are recovered,pooled and evaporated to give 410 mg (yield: 54%) of the expectedproduct.

Analysis (TLC on silica gel):

Rf=0.23 in CHCl₃ /MeOH/AcOH (10/3/1 v/v);

Rf=0.33 in CHCl₃ /MeOH/AcOH (5/3/1 v/v).

PREPARATION II

Preparation of L-THC-L-Pro-L-Arg-pNA.AcOH (Example 2)

Starting from L-THC-L-Pro-L-Arg-pNA.H-TFA obtained according toPreparation I, the product of Example 2, namelyL-THC-L-Pro-L-Arg-pNA.AcOH, is prepared by ion exchange on AMBERLITE®IRA 401 S resin (acetylated beforehand), the eluent being an MeOH/H₂ Omixture (3/2 v/v).

PREPARATION III

The product of Example 1 is prepared, the chromatography on silica gelin step Ie being replaced with HPLC (HYPERSIL® C 18 column of particlesize 3 micrometers). The product obtained is purer than that obtained inPreparation I.

What is claimed is:
 1. A tripeptide compound of the formula

    H-A.sub.1 -Pro-A.sub.3 -pNa

wherein A₁ is 2-(oxathiazolidin-4-yl)carbonyl (THC), and A₃ is Arg orLys, and acid addition salts thereof.
 2. A tripeptide compound accordingto claim 1 wherein (i) A₁ has the L- or D- configuration, and (ii) Proand A₃ have the L-configuration.
 3. A peptide compound according toclaim 2 which is selected from the group consisting ofH-L-THC-L-Pro-L-Arg-pNA and its addition salts.